The present invention relates to the field of molecular biology and the use of yeast for the production of ascorbic acid and ascorbic acid stereoisomers.
L-Ascorbic acid (Vitamin C, ASA) finds use in the pharmaceutical and food industry as a vitamin and antioxidant. The synthesis of ASA has received considerable attention over many years due to its relatively large market volume and high value as a specialty chemical. The Reichstein-Grussner method, a chemical route from glucose to ASA, was first disclosed in 1934 (Helv. Chim. Acta 17:311-328). Lazarus et al. (1989, xe2x80x9cVitamin C: Bioconversion via a Recombinant DNA Approachxe2x80x9d, Genetics and Molecular Biology of Industrial Microorganisms, American Society for Microbiology, Washington D.C. Edited by C. L. Hershberger) disclosed a bioconversion method for production of an intermediate of ASA, 2-keto-L-gulonic acid (2-KLG, KLG) which can be chemically converted to ASA. Saito et al. (1997, Applied and Environmental Microbiology, 63: 454-460) report on the construction of an expression system for the production of 2-KLG from D-sorbitol.
The presence of ASA in yeasts has been reported (Heick et al. Can. J. Microbiol., 1972, 18, 597-600) and the conversion of L-galactonic substrates to ASA in Candida yeast has been disclosed (U.S. Pat. Nos. 4,595,659, issued Jun. 17, 1986 and U.S. Pat. No. 4,916,068, issued Apr. 10, 1990). Costamagna et al. (Can. J. Microbiol., 1986, 32, 756-758) disclose the results of a study on ASA utilization by some yeasts. This report discloses that species of Cryptococcus and Candida were able to grow on ASA as well as iso-ascorbic acid.
In spite of the scientific advances made in the production of ASA and its biocatalytic intermediates, there remains a need for methods for the production of ascorbic acid in order to supply the world""s demand. The discovery of a method which utilizes a renewable carbon source to produce ascorbic acid would be particularly advantageous.
The present invention relates to the production of ascorbic acid or ascorbic acid stereoisomers in yeast. The present invention is based, in part, upon the unexpected discovery that multiple members of yeast which are able to grow on ascorbic acid or iso-ascorbic acid as a sole carbon source are capable of utilizing KLG as a sole carbon source to produce ascorbic acid.
Accordingly, the present invention provides methods for the production of ascorbic acid or an ascorbic acid stereoisomer from a yeast comprising the steps of obtaining a yeast capable of utilizing KLG to produce ascorbic acid or an ascorbic acid stereoisomer; and culturing the yeast in the presence of a carbon source under conditions suitable for the production of ascorbic acid or an ascorbic acid stereoisomer. ASA stereoisomers include D-ascorbic acid, D-araboascorbic acid and L-araboascorbic acid. The method may further comprise the step of recovering the ascorbic acid or ascorbic acid stereoisomer produced.
In one aspect of the present invention, the carbon source is a six carbon sugar acid. In another aspect of the present invention, the carbon source is a six carbon sugar and the yeast comprises either or both of a) a heterologous nucleic acid encoding an oxidative enzyme associated with the production of ascorbic acid or an ascorbic acid stereoisomer in said yeast and b) a heterologous nucleic acid encoding a reducing enzyme associated with the production of ascorbic acid or an ascorbic acid stereoisomer in said yeast.
In one embodiment of the present invention, the oxidative enzyme has a dehydrogenase activity. In another embodiment, the oxidative enzyme includes a glucose dehydrogenase activity, a gluconic acid dehydrogenase activity, a 2-keto-D-gluconic acid dehydrogenase activity, a galactose dehydrogenase activity, an L-sorbose dehydrogenase activity, an L-sorbosone-dehydrogenase activity, a 6 phosphogluconate kinase activity, a gluconate kinase activity, an L-idonic acid oxidase activity, and L-gulonic acid oxidase activity. In a further embodiment, the reducing enzyme is a reductase activity. In yet another embodiment, the reductase activity includes 2,5 DKG reductase activity, 2,3-DKG reductase, 5-keto reductase, 2-keto reductase and 2 ketogulonate reductase.
In one embodiment, the six carbon sugar acid includes 2-keto-L-gulonic acid, idonic acid, gluconic acid, 6-phosphogluconate, 2-keto-D-gluconic acid, 5-keto-D-gluconic acid, 2-ketogluconate-6-phosphate, 2, 5-diketo-L-gluconic acid, 2,3-L-diketogulonic acid, dehydroascorbic acid, erythroascorbic acid and D-mannonic acid. In another embodiment, the six carbon sugar includes glucose, gulose, sorbose, fructose, idose, galactose and mannose all in either D or L form.
In one embodiment of the present invention, the yeast is a member of the Imperfect yeast group. In another embodiment, the yeast is a member of the family Cryptococcaceae. In yet another embodiment, the yeast is Candida or Cryptococcus. In a further embodiment, the yeast is Candida blankii or Cryptococcus dimennae. 
In a preferred embodiment of the present invention, the yeast is Candida blankii or Cryptococcus dimennae, said carbon source comprises glucose, and the yeast comprises at least one of a heterologous oxidative enzyme and a heterologous 2,5-DKG reductase activity. In another preferred embodiment, the yeast is Candida blankii or Cryptococcus dimennae and said carbon source comprises D-sorbitol, L-sorbose or L-sorbosone, and the yeast comprises at least one of a D-sorbitol dehydrogenase activity, an L-sorbosone dehydrogenase activity, L-sorbose dehydrogenase activity and a galactose dehydrogenase activity.
In a preferred embodiment, the carbon source is glucose and the yeast comprises heterologous nucleic acid encoding at least one of (a) a glucose dehydrogenase (GDH) activity; (b) a gluconic acid dehydrogenase (GADH) activity; (c) a 2-keto-D-gluconic acid dehydrogenase (2-KDGDH) activity; and (d) a 2,5-diketo-D-gluconic acid reductase (2,5-DKGR) activity provided that if the yeast comprises heterologous nucleic acid for less than all of (a)-(d), then the yeast comprises endogenous nucleic acid such that the yeast comprises nucleic acid for each of (a)-(d) and is capable of converting glucose to ASA via the intermediate KLG.
The present invention also provides recombinant yeast capable of utilizing KLG to produce ascorbic acid or an ascorbic acid stereoisomer comprising either or both of a) a heterologous nucleic acid encoding an oxidative enzyme associated with the production of ascorbic acid or an ascorbic acid stereoisomer in said yeast and b) a heterologous nucleic acid encoding a reducing enzyme associated with the production of ascorbic acid or an ascorbic acid stereoisomer in said yeast.
In a preferred embodiment, the yeast is a member of the Imperfect Yeast group. In another preferred embodiment, the yeast is a member of the family Cryptococcaceae. In yet another preferred embodiment, the yeast is selected from members of the genera consisting of Candida and Cryptococcus, including Candida blankii and Cryptococcus dimennae. 
The present invention also encompasses a method for producing a recombinant yeast capable of utilizing a six carbon sugar to produce ASA or an ASA stereoisomer comprising the steps of obtaining a yeast capable of utilizing KLG to produce ASA or an ASA stereoisomer and introducing at least either or both of a) a heterologous nucleic acid encoding an oxidative enzyme associated with the production of ascorbic acid or an ascorbic acid stereoisomer in said yeast and b) a heterologous nucleic acid encoding a reducing enzyme associated with the production of ascorbic acid or an ascorbic acid stereoisomer in said yeast. In one embodiment of the method the yeast is a member of the Imperfect yeast group. In another embodiment, the yeast is a member of the family Cryptococcaceae, including Candida and Cryptococcus. In yet another embodiment, the yeast is Candida blankii. In a further embodiment, the yeast is Cryptococcus dimennae. In yet another embodiment, the yeast is Candida blankii or Cryptococcus dimennae, said carbon source comprises glucose and said yeast comprises a heterologous oxidative enzyme activity and a 2,5-DKGreductase activity. In an additional embodiment, the yeast is Candida blankii or Cryptococcus dimennae, said carbon source comprises D-sorbitol, L-sorbose or L-sorbosone, and said yeast comprises at least one of a D-sorbitol dehydrogenase activity, an L-sorbosone dehydrogenase activity, an L-sorbose dehydrogenase activity or a galactose dehydrogenase activity.